Competitive Exchange Diffusion to determine transport of hOCT2 inhibitors

Abstract

The organic cation (OC) transporter 2 (OCT2; SLC22A2) is the entry step of the OC secretory pathway of human renal proximal tubules. We used Competitive Exchange Diffusion (CED) to indirectly assess whether inhibitors of OCT2‐mediated transport also serve as substrates for transport. In CED, CHO cells that stably expressed hOCT2 were preloaded with [3H]MPP (‘M*’) to steady‐state (30 min). The solution was then replaced with one containing either (i) the pre‐loading solution (PLS), or (ii) PLS plus unlabeled test ligand. Whereas PLS alone supported no change in M* content over 5 min, presence of a transported ligand supported CED and a decrease in M* content. When the ligand was 100 uM unlabeled MPP (20× its Kt), M* decreased by 40% within 1 min. Saturating concentrations of the other OCT2 substrates (20× their Kt values), TEA, metformin and cimetidine supported M* efflux comparable to that supported by MPP (10 mM glucose, sucrose, or mannitol supported no CED). We then tested two compounds recently identified as strong inhibitors of OCT2 (Kido et al, J Med Chem, 54(13):4548–58, 2011): the sterol, adrenosterone (AS; IC50 of 6 uM) and the strong base, phenyltoloxamine (PT; IC50 of 6 uM). Interestingly, saturating AS or PT did not support CED‐induced efflux of M*. AS and PT both proved to be non‐competitive inhibitors of OCT2‐mediated MPP transport (decreasing Jmax with little or no change in Kt). NIH grant 5R01DK58251.